Bar-coding for Cytometry - How to multiplex samples to save reagents, costs and reduce batch effects
Bar-coding
What is it?
Staining individual samples in a unique way so that they can be identified for analysis even when mixed with other samples
- Bar-coding allows us to multiplex - mix our samples together.
- Stain and acquire ALL samples simultaneously
- BUT still analyze each sample individually.
Why BARCODE?
- Make work-flows more efficient
- Stain and process one large sample
- Can reduce acquisition time
- Can reduce cell loss
- Can reduce antibody requirements
- Improve data consistency
- By staining and processing all our samples together, we can
- Minimise batch effects
- Eliminate pipetting errors
- Eliminate carryover from sample to sample
- Provide additional and improved doublet discrimination
- Enables scaled-up experiments
- Reduces sample processing time
Why not BAR-CODE?
All of the benefits do not come for “free”
"While barcoding of samples has many benefits, it represents an additional step in the protocol, needs to be optimised on its own, and usually occupies cytometry channels which would otherwise be available to the measurement of target analytes."
Cossarizza et al. European Journal of Immunology, 47 (2017) 1584-1797
Why not BAR-CODE?
Integrating bar-codes into existing panels
- Bar-coding reagents occupy the same channels as our targets
- Bar-coding can have specific requirements like fixed cells
Limited number of barcodes
- 12-20 barcodes
- Fluorochrome selection
- Instrument capability
Spillover
- Bar-coding parameter spillover
How to Barcode
Each sample is tagged with a unique identifier.
In the case of flow cytometry, a specific dilution of one or two fluorescent dyes that react with all cells.
For mass cytometry we use several different approaches. We can use Palladium-tagged dyes or dilutions of pan-specific antibodies. In mass cytometry we can often use exclusive channels like the Palladium channels so that we don’t reduce the number of phenotypic antibodies we can use.
Current Barcoding Options
Flow Cytometry
There are 2 primary barcoding methods for flow cytoemtry samples:
- Becton-Dickinson provides the Violet Fluorescence Barcoding Kit
- BV421 and BV510 equivalent fluorophores are used (CBD 450 and CBD 500)
- Up to 16 samples can be barcoded
- kit is very easy to use
- NHS-Ester dyes tagged with fluorophores such as Alexa Fluor or Cyanine dyes (avaialble from many manufacturers)
- React with and stain ALL proteins on cells
- Combine 2 different dyes
- Can be titrated 3-5 times to provide 9-25 barcodes
Mass Cytometry
Palladium Bar-codes for universal cell multiplexing
Up to 20 samples can be barcoded using a 6 isotope - pick 3 format.
- Since Palladium is not traditionally used for antibody staining we don’t lose channels when we integrate this kit into our work flow.
- However, the kit only works only on fixed cells. You can choose when to bar-code though:
- At very beginning
- After surface stain
- Just before acquisition
Mass Cytometry
Live cell CD45-based bar-coding
Uses anti-CD45 antibodies and 7 Cadmium and 4 Platinum-conjugated antibodies in an
- 11 choose 3 (165 samples)
- or 7 choose 3 (35 samples Cadmium Only) format.
- Can be performed prior to marker staining.
- CD45 is insensitive to fixation and allows optimal detection of fixation sensitive epitopes.
- CD45 is not traditionally used for target markers so you can still use a large panel.
- If utilising the platinum channels will need to change live-dead to Rhodium or another marker.
Mass Cytometry
Universal live cell barcoding for human samples
The Bendall lab at Stanford has explored alternative ways of barcoding non-haematopoietic samples.
- Target ubiquitous molecules b2m and CD298
- Non lanthanide isotopes used for barcoding leaves all normal phenotyping channels open
- Unlike CD45, this approach is lineage independent so can be used for any cell types you need